粪便宏基因组来源低分子量碱性β-半乳糖苷酶的异源表达及酶学性质  

Heterologous expression and enzyme properties of low-molecular-weight and basic β-galactosidase derived from the metagenome of faeces

作　　者：范琴 曾璐瑶 杨金茹 韩楠玉 杨云娟[1,2,3] 黄遵锡 吴倩[1,2,3] 许波 FAN Qin;ZENG Luyao;YANG Jinru;HAN Nanyu;YANG Yunjuan;HUANG Zunxi;WU Qian;XU Bo(School of Life Sciences,Yunnan Normal University,Kunming 650500,Yunnan,China;Engineering Research Center of Sustainable Development and Utilization of Biomass Energy,Ministry of Education,Kunming 650500,Yunnan,China;Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment,Kunming 650500,Yunnan,China)

机构地区：[1]云南师范大学生命科学学院,云南昆明650500 [2]生物能源持续开发利用教育部工程研究中心,云南昆明650500 [3]云南省生物质能与环境生物技术重点实验室,云南昆明650500

出　　处：《微生物学通报》2022年第5期1594-1607,共14页Microbiology China

基　　金：国家自然科学基金(31860299);国家重点研发计划(2017YFB0308400)。

摘　　要：【背景】β-半乳糖苷酶在食品加工、临床医疗及基因工程等领域有重要的应用价值,开发酶活性高、热稳定性强的β-半乳糖苷酶已成为研究热点。【目的】从西黑冠长臂猿(Nomascus concolor)粪便微生物宏基因组中挖掘新型β-半乳糖苷酶并进行酶学性质研究。【方法】以西黑冠长臂猿粪便微生物宏基因组DNA为模板扩增β-半乳糖苷酶基因GalNC1-8,构建重组表达质粒pEASY-E2/GalNC1-8,转化至大肠杆菌(Escherichia coli)BL21(DE3)异源表达,研究其酶学性质。【结果】获得GH35家族碱性β-半乳糖苷酶GalNC1-8,其分子量为28.18 kDa,最适温度为50℃,最适pH为8.0。将该酶在30−50℃下处理1 h,剩余酶活仍保持在80%以上;pH 7.0−9.0下处理1 h,剩余酶活大于54%。在含乙醇的反应体系中,其酶活性几乎不受影响;β-巯基乙醇、丙三醇、甲醇、Na^(+)、K^(+)和Li^(+)对其酶活性有促进作用。在0.5−3.5 mol/L NaCl下处理1 h后,仍保留50%以上的酶活性。【结论】从粪便微生物宏基因组中克隆得到β-半乳糖苷酶基因GalNC1-8,并在E.coli BL21(DE3)中成功表达。GalNC1-8是已知宏基因组来源蛋白质分子量最小的β-半乳糖苷酶,其pH作用范围较广、温度稳定性和NaCl耐受性较好,具有较好的应用前景。[Background]β-galactosidase has important application value in many fields such as food processing,clinical medicine,and genetic engineering.Thus,the development ofβ-galactosidase with high activity and strong thermal stability has become a research focus.[Objective]This paper aims to screen novelβ-galactosidases from the fecal microbial metagenome of Nomascus concolor and study its enzymatic properties.[Methods]β-galactosidase gene GalNC1-8 was cloned by PCR with the fecal microbial metagenomic DNA of N.concolor as template.Then,the recombinant plasmid pEASY-E2/GalNC1-8 was constructed and transformed into Escherichia coli BL21(DE3)for expression,and the enzymatic properties of recombinase GalNC1-8 was tested.[Results]The basicβ-galactosidase GalNC1-8,belonging to GH35 family,had the molecular weight of 28.18 kDa.The optimal conditions for GalNC1-8 were pH 8.0 and 50°C.After incubation at 30,37,40,and 50℃ for 1 h,the relative enzyme activity was still above 80%.After 1 h treatment at pH 7.0−9.0,the enzyme kept more than 54%of the activity.The activity of GalNC1-8 was hardly influenced by the reaction system containing ethanol.β-mercaptoethanol,glycerol,methanol,Na^(+),K^(+),and Li^(+) enhanced the enzyme activity.It retained above 50%activity after being treated with 0.5−3.5 mol/L NaCl at 50℃ for 1 h.[Conclusion]A novel β-galactosidase gene GalNC1-8 was screened from the N.concolor fecal microbial metagenome,which expressed in E.coli BL21(DE3).GalNC1-8 has the lowest molecular weight among the known metagenome-derivedβ-galactosidases.With adaptability to a wide pH range,thermal stability,and salt tolerance,it has good application prospects.

关 键 词：Β-半乳糖苷酶 粪便微生物 宏基因组 酶学性质 

分 类 号：Q78[生物学—分子生物学] Q814.9